Definition of a New HLA B*52-Restricted Rev CTL Epitope Targeted by an HIV-1-Infected Controller

The analysis of T-cell responses in HIV-1-infected controllers may contribute to a better understanding of the protective components of the immune system. Here, we analyzed the HIV-1-specific T-cell response in a 59-year-old HIV-1-infected controller, infected for at least seven years, who presented with low viral loads ranging from <20 copies/mL to 200 copies/mL and normal CD4 counts of >800 cells/µL. In γ-IFN-ELISpot assays using freshly isolated PBMCs, he displayed a very strong polyclonal T-cell response to eight epitopes in Gag, Nef and Rev; with the dominant responses directed against the HLA-B*57-epitope AISPRTLNAW and against a so-far-unknown epitope within Rev. Further analyses using peptide-stimulated T-cell lines in γ-IFN-ELISpot assays delineated the peptide RQRQIRSI (Rev-RI8) as a newly defined HLA-B*52-restricted epitope located within a functionally important region of Rev. Peptide-stimulation assays in 15 HLA-B*52-positive HIV-1-infected subjects, including the controller, demonstrated recognition of the Rev-RI8 epitope in 6/15 subjects. CD4 counts before the start of antiviral therapy were significantly higher in subjects with recognition of the Rev-RI8 epitope. Targeting of the Rev-RI8 epitope in Rev by CTL could contribute to the positive association of HLA-B*52 with a more favorable course of HIV-1-infection.


Introduction
HIV-1-specific cytotoxic T-lymphocytes (CTL) play an important role in the control of HIV-1. The emergence of CTL was associated with suppression of HIV-1 replication at primary HIV-1-infection [1] and the frequency of HIV-1-specific CTL correlated to a lower plasma viremia in the chronic phase of HIV-1-infection [2]. However, despite frequent detection of HIV-1-specific CTL in the asymptomatic stage of HIV-1 infection, without antiviral therapy most HIV-1-infected patients develop progressive immunodeficiency. Only a small group of subjects, the long-term non-progressors, maintain normal CD4 counts and low viral loads for more than ten years, and only a few subjects, the elite controllers, are able to suppress viral replication below 50 copies/mL [3]. The reasons for the long-term control of HIV-1-replication are not fully defined and may vary between individual controllers. Strong HIV-1-specific CTL responses have been observed in at least a subgroup of long-term non-progressors (4). The importance of CTL-mediated control is underlined by the strong correlation of certain HLA-I alleles, such as HLA B*57, B*27, B*52 or B*14, with a more favorable course of HIV-1 infection [4][5][6]. It has been

Study Subjects
The 59-year-old controller (subject #1) was diagnosed with HIV-1 infection when he was treated in the hospital for recurrent epileptic seizures. Since an ischemic stroke seven years previously he had suffered from hemiparesis of his right leg and right arm. He was a smoker with hypertonic blood pressure. The time point of HIV-1 infection is not known but was established to have occurred prior to his severe neurological disability.
At diagnosis, he displayed a CD4 count of 1004 cells/µL and HIV-1-specific antibodies measured by ELISA (ARCHITECT HIV Ag/Ab Combo Assay, Abbott, Wiesbaden, Germany) and immunoblot (Geenius HIV 1/2 Confirmatory Assay, Bio-Rad laboratories, Feldkirchen, Germany). He maintained normal CD4 counts >800 cells/µL over the next 453 days post-diagnosis (Table 1). His viral load measured by real-time HIV-1 PCR (Abbott RealTime HIV-1 assay, Abbott, Wiesbaden) was 40 copies/mL at diagnosis. with subsequent low viral loads ranging between <20 and 20 copies/mL until day 293 post-diagnosis (Table 1). At day 383, four weeks after a traumatic subarachnoid hemorrhage and fracture of his right humerus, he displayed a transient increase of viral load to 250 copies/mL with spontaneous decline to <20 copies/mL at day 453. A resistance analysis from plasma obtained at day 383 post-diagnosis revealed the presence of several mutations in reverse transcriptase (RT, 41L, 210W, 215A) and protease (33F, 43T, 46L, 53L, 82A, 88D) which were associated with high-level resistance against zidovudine, stavudine and several protease inhibitors. This indicated the transmission of a drug-resistant virus as the patient has not been treated with antiretroviral drugs in the past.
In addition to the controller (subject #1), we investigated 14 HLA-B*52-positive, HIV-1-infected patients (clinical characteristics shown in Table S1). All were on antiretroviral combination therapy (cART) for a median time of 75 months (range 3-315 months). They presented with a current median viral load of <20 copies/mL (range: <20 to 40 copies/mL) and a current median CD4 count of 872 (range 351-1434).

Isolation of PBMCs
PBMCs were isolated from HIV-1-infected patients by density-gradient centrifugation, in Leucosep tubes (Greiner Bio One GmbH, Frickenhausen, Germany) pre-filled with 15 mL of Lymphoflot separation medium (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). The tubes were filled with a maximum volume of 30 mL citrate blood diluted with PBS to 35 mL, then centrifuged 20 min at 760× g and RT. The supernatant was collected and centrifuged 10 min at 610× g and RT. The resulting cell pellet was washed once in 30 mL PBS and then resuspended in appropriate culture medium.
For ELISpot culture assay, 50 µL of T-cell suspension was added to 50 µL R5AB medium in each well of a coated plate. Then, 2 µL of dissolved peptides were added directly into the wells to a final concentration of 20 µg/mL. Respective cell suspensions without peptides served as negative controls. The plates were incubated for 18 to 40 h at 37 • C and 7% CO 2 .
For color development, the plates were first washed six times with PBS containing 0.05% Tween-20 (PBS-T0.05%), then incubated with 100 µL/well biotinylated anti-human γ-IFN monoclonal antibody 7-B6-1 (Mabtech, Stockholm, Sweden) at a final concentration of 2 µg/mL for two hours at room temperature. Afterwards, the plates were washed again with PBS-T0.05% six times, then incubated with 100 µL/well avidin/peroxidase substrate (Vectastain ® Elite ® ABC-Kit, Linaris, Wertheim, Germany) for one to three hours at room temperature, then washed again three times with PBS-T0.05% and three times with PBS. Finally, 100 µL AEC substrate (3-amino-9-ethylcarbazole, Sigma-Aldrich, Steinheim, Germany) containing 0.06% H 2 O 2 was added as chromogen to each well. The spots developed within ten minutes, and the reaction was stopped by washing the plates three times with distilled water. The plates were air-dried, and the spots were counted using an ELISpot reader (AID, Strassberg, Germany). Results are reported as SFUs (spot-forming units) either per 50 µL or per cell number.
A peptide-specific response was defined as positive if the number of spot-forming units (SFUs) exceeded the following thresholds: ≥10 SFUs and >2 fold over background (SFUs without peptide).
To assess the functional avidity of peptides by peptide titration assay, peptides were added in serial dilutions ranging from 20 µg/mL to 1 ng/mL to ELISpot plates and incubated with T-cell lines (1 × 10 5 per well in duplicates) for 22-36 h.

HLA Typing
HLA class-I typing was performed using standard serological techniques (Biotest AG, Dreieich, Germany) or genotypic analyses (enzyme-linked probe hybridization assay Biotest ELPHA, Biotest AG) according to the manufacturer's guidelines.

HLA-Restriction Analysis
HLA-I-/II-restriction was determined by ELISpot assays using antibodies blocking either CD4 or CD8. The cells were incubated at a final concentration of 50 µg/mL of an anti-CD4 (OriGene Technologies, Rockville, Maryland, USA) or 625 ng/mL of an anti-CD8 antibody (BD Bioscience, San Jose, CA, USA) for one hour at 37 • C and 7% CO 2 . Then, peptides were added directly to the wells at a final concentration of 20 µg/mL. The restricting HLA-I allele was determined using HLA-matched allogeneic B-LCLs from HLA-I-typed blood donors. These target cells were incubated with the respective peptides for one hour and washed twice with 15 mL PBS. Peptide-sensitized target cells and target cells without peptides were then co-incubated with the T-cell lines in a γ-IFN ELISpot assay, using 2 × 10 5 target cells per well and 2 × 10 5 T-cells in duplicate.

Statistical Analysis
The statistical analyses were carried out using GraphPad Prism 8.0. (GraphPad Soft-162 ware, San Diego, CA, USA).

Definition of the Rev-RI8 Peptide as a New Epitope in Rev
To define the minimal epitope within Rev07-11, peptide-stimulated T-cell lines were analyzed for recognition of 15 amino acid long peptides overlapping with the Rev07-11 by 11 amino acids. Recognition of the peptides Rev07-11 (RRRRWRERQRQIRSI and Rev07-12 (WRERQRQIRSISGWI) with lack of recognition of the peptides Rev07-13 (QRQIRSISG-WILSTY) and Rev07-10 (ARRNRRRRWRERQRQ) indicated that the epitope was located with the sequence WRERQRQIRSI (Figure 2a). The fine specificity of CTL was then mapped by a set of overlapping short peptides of 8 to 10 amino acids revealing the peptide Rev-RI8 (RQRQIRSI) as the minimal epitope (Figure 2b). Omission of the arginine at the first position or the isoleucine at the C-terminal end abrogated recognition (Figure 2b).

Peptide Rev-RI8 Is Presented by HLA B*52
To confirm that the observed γ-IFN responses to the rev peptide Rev07-11 with minimal epitope Rev-RI8 are mediated by CD8+ T-cells and not by CD4+ T-cells, we next performed a peptide-recognition assay using the controller's T-cell lines in the presence of anti-CD4 or anti-CD8 antibodies. Recognition of peptide Rev-RI8 in γ-IFN ELISpot assays could be blocked by anti-CD8-antibodies but not by anti-CD4-antibodies, demonstrating presentation of the Rev-RI8 epitope by HLA-I molecules (Figure 3a). HLA-restriction of Rev-RI8 was analyzed in γ-IFN-ELISpot assays using Rev-RI8 -specific CTL lines and peptide-sensitized allogenic EBV generated B-LCL sharing only one HLA-1-allele each

Definition of the Rev-RI8 Peptide as a New Epitope in Rev
To define the minimal epitope within Rev07-11, peptide-stimulated T-cell line analyzed for recognition of 15 amino-acid-long peptides overlapping with the Re by 11 amino acids. Recognition of the peptides Rev07-11 (RRRRWRERQRQIRS Rev07-12 (WRERQRQIRSISGWI) with lack of recognition of the peptides Re

Peptide Rev-RI8 Is Presented by HLA B*52
To confirm that the observed γ-IFN responses to the rev peptide Rev07-11 with m imal epitope Rev-RI8 are mediated by CD8+ T-cells and not by CD4+ T-cells, we next p formed a peptide-recognition assay using the controller's T-cell lines in the presence anti-CD4 or anti-CD8 antibodies. Recognition of peptide Rev-RI8 in γ-IFN ELISpot assa could be blocked by anti-CD8-antibodies but not by anti-CD4-antibodies, demonstrati presentation of the Rev-RI8 epitope by HLA-I molecules (Figure 3a). HLA-restriction Rev-RI8 was analyzed in γ-IFN-ELISpot assays using Rev-RI8 -specific CTL lines and pe tide-sensitized allogenic EBV generated B-LCL sharing only one HLA-1-allele each w The sensitizing activity of peptide Rev-RI8 was determined by incubation of serial dilutions of the Rev-RI8 peptide with the Rev-RI8-specific T-cell lines from three HLA-B*52-positive subjects in IFN-γ ELISpot assays. The 50% peptide-sensitizing activity was determined as 2.3 µg/mL with loss of peptide recognition at 10 ng/mL (Figure 4).  The sensitizing activity of peptide Rev-RI8 was determined by incubation of serial dilutions of the Rev-RI8 peptide with the Rev-RI8-specific T-cell lines from three HLA-B*52-positive subjects in IFN-γ ELISpot assays. The 50% peptide-sensitizing activity was determined as 2.3 µg/mL with loss of peptide recognition at 10 ng/mL (Figure 4).   The sensitizing activity of peptide Rev-RI8 was determined by incubation of serial dilutions of the Rev-RI8 peptide with the Rev-RI8-specific T-cell lines from three HLA-B*52-positive subjects in IFN-γ ELISpot assays. The 50% peptide-sensitizing activity was determined as 2.3 µg/mL with loss of peptide recognition at 10 ng/mL (Figure 4).  HIV-1-specific CTL, the prevalence of Rev-RI8-specific T-cells was assessed by stimulation of PBMCs with peptide Rev-RI8 and analysis of outgrowing cells after 10-14 days in γ-IFN -ELISpot assays. Rev-RI8-specific T-cells could be detected in 6 out of 15 HLA B*52-positive patients (40%) including controller #1 ( Figure 5, Table S2). Subjects with recognition of Rev-RI8 displayed significantly higher pre-ART CD4+ T-cells ( Figure 6, p = 0.012), whereas there were no differences regarding current CD4 counts, current and pre-ART viral loads, current and pre-ART CD8 counts, or time on antiretroviral therapy (Table S2). The higher pre-ART CD4 counts of Rev-RI8 responders was significant, even when omitting controller #1 from the analysis (p = 0.0290).

Recognition of Rev-RI8 in a Cohort of HLA-B52-Positive HIV-1-Infected Patients
To investigate how frequently the Rev-RI8 epitope is targeted by HLA-B*52-positive HIV-1-infected patients, recognition of the Rev-RI8 was assessed in an additional 14 HLA-B*52-positive HIV-1-infected subjects of the Erlangen HIV cohort. As all patients were on an effective antiretroviral therapy, which is associated with a decline of circulating HIV-1-specific CTL, the prevalence of Rev-RI8-specific T-cells was assessed by stimulation of PBMCs with peptide Rev-RI8 and analysis of outgrowing cells after 10-14 days in γ-IFN -ELISpot assays. Rev-RI8-specific T-cells could be detected in 6 out of 15 HLA B52-positive patients (40%) including controller #1 ( Figure 5, Table S2). Subjects with recognition of Rev-RI8 displayed significantly higher pre-ART CD4+ T-cells ( Figure 6, p = 0.012), whereas there were no differences regarding current CD4 counts, current and pre-ART viral loads, current and pre-ART CD8 counts, or time on antiretroviral therapy ( Table S2). The higher pre-ART CD4 counts of Rev-RI8 responders was significant, even when omitting controller #1 from the analysis (p = 0.0290).

Discussion
In this study, we identified an HIV-1-infected controller with a very strong polyclonal CTL response targeting at least eight different CTL epitopes including a newly defined

Discussion
In this study, we identified an HIV-1-infected controller with a very strong polyclonal CTL response targeting at least eight different CTL epitopes including a newly defined HLA-B*52-restricted CTL epitope in Rev. Strong CTL responses have previously been demonstrated at least in subsets of HIV-1-infected controllers supporting an important role of CTL in the control of HIV-1 [15]. The HLA-B*57+ controller #1 recognized at least four HLA B*57-restricted CTL epitopes, three in Gag and one in Nef, with the highest CTL frequency targeting the HLA-B*57-restricted Gag epitope AW10. This is in line with reports by other groups that HLA-B*57 is associated with a better suppression of HIV-1 replication and a more favorable course of HIV-1-infection [4,6,16,17]. In HLA B*57+ subjects, the Gag epitopes KF11, AW10 and TW10 are usually immunodominant epitopes targeted by higher CTL frequencies than other epitopes restricted by other HLA alleles [4].
Interestingly, we observed in controller #1 very high CTL frequencies targeting the newly defined HLA-B*52-restricted CTL epitope Rev-RI8 in Rev at a similar magnitude as the immunodominant HLA B*57-epitope AW10. With a glutamine at the P2 position and an isoleucine at the C-terminal position, the Rev-RI8 epitope corresponds to the published peptide-binding motif of HLA B*52-restricted epitopes [18]. While there is sequence variation within the epitope, the P2 and P8 anchor positions are highly conserved (Tables S3 and S4) [19]. HLA-B*52 has been associated with resistance to HIV-1-infection [20], with early control after infection [16] and with non-progression to AIDS [21][22][23][24][25]. So far, it has been speculated that this beneficial effect of HLA-B*52 is mediated by CTL selecting escape mutations within HLA B*52-restricted Gag-and Pol-epitopes leading to decreased viral replicative capacity [22,26]. Here, we report on the delineation of the Rev-RI8 peptide as a new HLA-B*52-restricted CTL epitope in Rev. The Rev-RI8 epitope is located in a functionally important sequence region in Rev [27]. The first three amino acids of the Rev-RI8 epitope are part of a sequence domain that mediates both the nuclear localization and the binding of Rev to the Rev-responding element. The amino acids at positions P5 to P8 are required for multimerization of Rev. Thus, we could hypothesize that CTL escape mutations within Rev-RI8 could affect Rev function. However, due to the low viral load, we were not able to obtain autologous Rev sequences from controller #1.
Recognition of the Rev-RI8 epitope was significantly associated with higher pre-ART CD4 counts in our cohort of HLA-B*52+ patients indicating a beneficial role of Rev-RI8specific CTL. This is in line with another study reporting on a slower progression to AIDS in subjects with detection of Rev-specific CTL [24]. In contrast, two other studies did not find a statistical correlation between the magnitude or breadth of Rev-specific CTL and the control of HIV-1-replication [28,29], but HLA-B*52 positivity and HLA-B*52-specific CTL responses were not reported in both studies. This suggests that beneficial effects of Rev-specific CTL do not depend on recognition of Rev per se, but on targeting mutationally constrained CTL epitopes in which escape mutations lead to a lower fitness of the virus [8].
To understand the relative contribution of recognition of the Rev-RI8 epitope for the control of HIV-1, further studies are needed to analyze the effect of mutations within the Rev-RI8 epitope both on CTL recognition and on Rev function.
The patient displayed several drug-resistance mutations within RT and protease demonstrating infection with a drug-resistant virus as the patient had never received antiretroviral therapy. As several of the observed drug-resistance mutations could impair viral replication capacity [30,31], we hypothesize that the transmission of an attenuated drug-resistant virus could have contributed to the long-term control of HIV-1 in this controller. This is supported by the observations of another group reporting on frequent detection of drug-resistant viruses in plasma viral sequences in individuals who became HIV controllers [32].
It has been shown that Gag-specific CTL epitopes are frequently immunodominant and that targeting Gag was associated with control [33,34]. Furthermore, it has been speculated that this beneficial role of Gag responses could be attributed to high abundance of Gag and Gag-derived peptides in infected cells [35,36], to mutational constraints preventing rapid CTL escape [8,9] and the ability of Gag-specific CD8+ T cells to target incoming virions, thus being able to eliminate infected cells prior to production of virions [37]. Although the HLA-B*57 epitopes in Gag are conserved, HIV-1 can develop escape mutations within these epitopes [38,39] and compensate the fitness costs of these mutations by introduction of compensatory mutations outside of the epitopes [40,41]. Therefore, targeting of multiple CTL epitopes in functionally important viral sequences seems to be decisive to lower the risk of immune evasion by escape mutations [42]. In this context, it is remarkable that the controller targeted the HLA-B*52-restricted Rev-RI8 epitope with similar CTL frequency as the HLA-B*57-restricted Gag-epitopes suggesting a highly efficient presentation of the Rev-RI8 epitope. As Rev is expressed early in the viral replication cycle, recognition of this immunogenic Rev epitope could have been an important factor in the long-term control of HIV-1 in this controller. Further studies are needed to investigate whether inclusion of the Rev-RI8 Rev epitope into vaccines could improve CTL-mediated control of HIV-1 in HLA-B*52-positive subjects.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Medical Faculty of the Friedrich-Alexander-Universität Erlangen-Nürnberg (Number 250_15B, 6.7.2020).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.